The overall object of the proposed research is to study the functional behavior of hemoglobin, with particular emphasis on the mechanism of ligation reactions. Human variants: Hb Sydney Val E11 (G7) yields Ala; Hb Zurich His Beta(63) yields Arg; Hb Hammersmith Beta(42) Phe yields Ser; Hb Bryn Mawr Phe Beta(85) yields Ser; fetal hemoglobins (FI, FII); the minor components of normal adult hemoglobin (AIa, AIb, AIc) and cat hemoglobins have been selected for the present study. The kinetic studies will be carried out through the use of rapid mixing and relaxation techniques. The human mutant hemoglobins listed above are unstable and precipitate as Heinz bodies in circulating red blood cells, resulting in their premature hemolysis. They all involve substitutions of invariant residues. The studies planned here will provide information regarding the specific role of these residues in ligation reactions of hemoglobin. The minor components and fetal hemoglobin are found at elevated levels in diabetic and sickle cell patients, respectively. These components will be isolated, characterized and their equilibrium and kinetic behavior in ligation reactions will be studied. Hemoglobins FI, AIc and Hb B (cat), with the N-terminals of their Beta chains blocked, constitute an interesting group for studying the role of beta chain N-terminals in generating the kinetic heterogeneity of alpha and beta chains in the hemoglobin tetramer and their interaction with inorganic phosphates.